Tumor necrosis factor-α (TNFα) is a key regulator of inflammatory responses and has been implicated in many pathological conditions, such as rheumatoid arthritis, inflammatory bowel disease, psoriatic arthritis, vasculitis, ankylosing spondylitis, and juvenile chronic arthritis.
TNFα is present on the cell surface as a homotrimeric protein in which each subunit is initially translated as a 26 kD type II transmembrane precursor protein by cells of the immune system, including macrophages and monocytes. After being cleaved at a site proximal to the transmembrane domain of TNFα by TNFα converting enzyme (TACE), a soluble trimeric form of TNFα (17 kD) is released into the blood and exerts its activity by binding to two structurally distinct type I and type II TNF receptors (TNFRI and TNFRII) on effector cells.
The transmembrane form of TNFα plays a dual role in transmitting signals as a ligand and as a receptor which relays signals back to the cell. Therefore, transmembrane TNFα plays an important role in local inflammation in a cell-to-cell contact manner.
Anti-TNFα agents, including infliximab, adalimumab, etanercept and certolizumab pegol, bind to transmembrane TNFα on transmembrane TNFα-transfected cells with similar binding affinities, but their binding affinities are weaker than for soluble TNFα (Kaymakcalan, Z., P. Sakorafas, et al. (2009). “Comparisons of affinities, avidities, and complement activation of adalimumab, infliximab, and etanercept in binding to soluble and membrane tumor necrosis factor.” Clin. Immunol. 131(2): 308-316.). Previous reports indicated that infliximab, adalimumab and etanercept similarly bind to transmembrane TNFα on TNFα producing cells, and infliximab and adalimumab (two monoclonal antibodies) seem to transmit stronger inhibitory signals through transmembrane TNFα than etanercept (Nesbitt, A., G. Fossati, et al. (2007). “Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents.” Inflamm Bowel Dis 13(11): 1323-1332.). The binding effects of these antagonists on the transmembrane form of TNFα are different, and may cause different results on clinical diseases (Taylor, P. C. (2010). “Pharmacology of TNF blockade in rheumatoid arthritis and other chronic inflammatory diseases.” Curr Opin Pharmacol 10(3): 308-315.).
Unlike anti-TNFα antibodies, etanercept is not clinically effective for the pathogenesis of granulomatous diseases, in which the transmembrane TNFα may play a critical role (Mitoma, H., T. Horiuchi, et al. (2008). “Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor alpha-expressing cells: comparison among infliximab, etanercept, and adalimumab.” Arthritis Rheum 58(5): 1248-1257.). Etanercept is a dimeric molecule composed of the extracellular domain of TNF receptor 2 (p75 TNF receptor) and the Fc fragment of human IgG1. It is currently being used for the treatment of rheumatoid arthritis. However, 25% to 38% of patients show no response. This is suspected to be partially due to insufficient affinity of this protein to TNFα. The bivalent etanercept molecule forms a 1:1 complex with the TNFα trimer in which two of the three receptor binding sites on TNFα are occupied by etanercept, and the third receptor binding site is open (Scallon, B., A. Cai, et al. (2002). “Binding and functional comparisons of two types of tumor necrosis factor antagonists.” J Pharmacol Exp Ther 301(2): 418-426.). Cells expressing transmembrane TNFα that bind etanercept are not lysed in vitro in the presence or absence of complement (Arora, T., R. Padaki, et al. “Differences in binding and effector functions between classes of TNF antagonists.” Cytokine 45(2): 124-131. (2009).). Previous reports show that etanercept exhibits a relative low affinity toward the transmembrane TNFα as compared with infliximab. It is hypothesized that the induction of apoptosis by high-affinity TNFα binding agents such as sTNFR1 or anti-TNFα antibody infliximab is due to ligation of transmembrane TNFα and not to the neutralization of secreted TNFα, which can be a survival factor for monocytic cells. Therefore, enhancement of the binding strength of the bivalent etanercept to transmembrane TNFα may be a solution for increasing the efficacy in the treatment of both rheumatoid arthritis and, possibly, Crohn's disease.
Functional affinity (avidity) is a measure of the overall binding strength of an antigen with many antigenic determinants and multivalent antibodies. Polymerization of antigen-binding partners greatly increases their availability (or valency) for binding to a group of specific identical ligands in very close proximity to a target cell. TNFα family receptors form homotrimers when bound to their cognate ligands. The effect of oligomerization of soluble chimeric receptors on their affinity to their ligands has been studied. It was found that the best results were not obtained with a trimer, as expected, but with pentamers. Trimers are as efficient as dimers, but five times less efficient than the pentamers (Holler, N., T. Kataoka, et al. (2000). “Development of improved soluble inhibitors of FasL and CD40L based on oligomerized receptors.” J Immunol Methods 237(1-2): 159-173.).
Trivalent assembly of a heterologous target-binding domain by using a trimerization domain has been reported. Examples of trimerizing domains include C-propeptide of procollagens, coiled-coil neck domain of collectin family proteins, C-terminal portion of FasL and bacteriophage T4 fibritin foldon domain (Hoppe, H. J., P. N. Barlow, et al. (1994). “A parallel three stranded alpha-helical bundle at the nucleation site of collagen triple-helix formation.” FEBS Lett 344(2-3): 191-195.; Frank, S., R. A. Kammerer, et al. (2001). “Stabilization of short collagen-like triple helices by protein engineering.” J Mol Biol 308(5): 1081-1089; Holler, N., A. Tardivel, et al. (2003). “Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.” Mol Cell Biol 23(4): 1428-1440.).
The target binding domain of these trimerized heterologous target-binding protein assemblies can be protein hormones, cytokines, lymphokines, growth factors, lectins, enzymes and soluble receptor fragments; or adhesion molecules, such as selectins and integrins.
A short alpha-helical collagen-like peptide capable of self-trimerization and propagation of the heterologous fusion proteins from either the C- or N-terminal direction has been reported in EP1798240B1. In comparison with the immunoglobulin G (IgG) molecules, there are disadvantages with these trimeric fusion molecules when they are used for therapeutic applications. The disadvantages include: (1) Downstream process—unlike immunoglobulin G (IgG) molecules which can be easily purified by affinity chromatographies on protein A or G-conjugated resins through binding to the Fc fragment of IgG, resulting in more than 98% in homogeneity of the product at the first step of purification scheme, purification of the above fusion proteins for therapeutic applications is a challenge since no commercial affinity columns are available; (2) Low serum half-life—the Fc fragment of the IgG molecule has an increased systemic half-life resulting from the binding of Fc to the neonatal Fc receptor (FcRn), which is present in endothelial cells that line blood vessels. Upon binding to FcRn, IgG is protected from degradation and re-cycled into circulation, keeping the molecule in circulation longer. The pharmacokinetic properties of these trimeric fusion proteins have been improved as the Fc fragment binds to the FcRn and is responsible for maintaining the long half-life of trimeric fusion proteins in circulation.
It is possible to introduce an Fc fragment to one end of the trimeric molecule to become a trimeric Fc fusion protein. It is speculated that such a trimeric Fc fusion protein can be purified more efficiently using protein A-conjugated resins; most importantly, it may confer a longer plasma half-life, resolving both purification and pharmacokinetics issues. Methods to generate trimeric Fc fusion proteins have been described by fusion of an Fc fragment with different TNF homology domains in an N-terminal to C-terminal direction and then expressed in mammalian cells as secretory fusion proteins (Muller, N., A. Wyzgol, et al. (2008). “Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization.” Febs J 275(9): 2296-2304. Wyzgol, A., N. Muller, et al. (2009). “Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand.” J Immunol 183(3): 1851-1861.). The TNF homology domain (THD) is located at the C-terminus of the TNF ligand family, and is responsible for trimerization of TNF ligands and the binding of their cognate receptors. The results indicated that when Fc fused with different THDs, the dimerization force of the Fc domain and the trimerization force of the different THD compete each other, resulting in different oligomerization patterns of dimer, trimer or hexamers. The production of homogeneous trimeric or hexameric Fc-THD fusions was hampered by the intrinsic low trimerization capability of the THD and a second trimeric coiled-coil domain of tenascin-C (TNC) was introduced in-between the Fc and THD domains to stabilize the homo-oligomeric structure. U.S. Patent Application Publication U.S. 2005/0255547 described that a hexameric polypeptide might be assembled by fusion of an extracellular domain of a TNF receptor family protein with a hexameric moiety, wherein the hexameric moiety can be either a “true” hexamer, or a combination of “dimer of trimer” or “trimer of dimer”. Unfortunately, no examples are available to demonstrate the assembly of such a stable hexameric structure. In order to obtain a predominantly trimeric or/and hexameric Fc-containing fusion molecule, a novel trimerizing domain is needed to drive and stabilize the trimeric assembly of the fusion partners when a stable dimerizing Fc fragment is present.
The sequence Gly-Pro-Hyp is the most stable and common triplet in collagen and the peptide (Gly-Pro-Hyp)10 (SEQ ID NO: 33) can self-associate into a highly stable triple helical structure in vitro (Chopra, R. K. and V. S. Ananthanarayanan (1982). “Conformational implications of enzymatic proline hydroxylation in collagen.” Proc Natl Acad Sci USA 79(23): 7180-7184; Yang, W., V. C. Chan, et al. (1997). “Gly-Pro-Arg confers stability similar to Gly-Pro-Hyp in the collagen triple-helix of host-guest peptides.” J Biol Chem 272(46): 28837-28840). Previously, a short collagen-like peptide (Gly-Pro-Pro)10 (SEQ ID NO: 21) was adopted to drive the trimerization of its monomeric fusion partners by expression of the recombinant cDNA construct in a mammalian system (Fan, C. Y., C. C. Huang, et al. (2008). “Production of multivalent protein binders using a self-trimerizing collagen-like peptide scaffold.” Faseb J 22(11): 3795-3804.). It is not known whether (Gly-Pro-Pro)10 (SEQ ID NO: 21) is still capable of initiating trimeric assembly of its fusion partners in mammalian cells when a stable dimerization domain, such as the IgG Fc fragment, is introduced at either the N- or C-terminal end.
Thus, there is a need for a TNFα inhibiting molecule which is stable in circulation, binds to transmembrane TNFα with suitable avidity to be effective, and which forms a stable trimer or hexamer structure, even in the presence of protein domains which tend to form dimers.